This invention relates to enhancing the ability of oligonucleotides, such as antisense oligonucleotides and ribozymes, to inhibit protein production, cell proliferation, and/or the multiplication of a foreign organism.
Oligonucleotides such as antisense oligonucleotides and ribozymes can hybridize to a target RNA, such as mRNA, and inhibit production of protein from that RNA. Numerous mechanisms have been proposed to explain the effects of antisense oligonucleotides. For example, see Helene, C. and Toulme, J. Biochimica et Biophysica Acta 1049:99 (1990), and Uhlmann, E. and Peyman, A. Chemical Reviews 90:543 (1990). Proposed mechanisms include formation of a DNA:RNA substrate for cellular RNAse H, which degrades the RNA strand involved in the duplex; hybridization of an antisense oligonucleotide to nascent mRNA leading to premature transcription termination; and interfering with mRNA processing by hybridizing to a pre-mRNA intron/exon junction. These and several other proposed mechanisms for inhibiting nucleic acid activity by antisense oligonucleotides are based upon the ability of antisense oligonucleotides to hybridize to a target RNA sequence.
Oligonucleotides can also inhibit viruses by a sequence non-specific mechanism. For example, Majumdar, et al., Biochemistry 28:1340 (1989) describe the use of phosphorothioate oligonucleotides to inhibit HIV reverse transcriptase.
Hybridization of an oligonucleotide to a target nucleic acid sequence occurs by hydrogen bonding between complementary nucleotides present on the oligonucleotide and the nucleic acid target site. If the individual nucleotides on the target nucleic acid sequence are not accessible to the oligonucleotide, hybridization will be prevented. Inaccessibility of nucleotides can be due to various factors such as secondary structure of the target nucleic acid and proteins associated with target nucleic acid.
Hogan, et al., U.S. Pat. No. 5,030,557, describe the use of a "helper" oligonucleotide to help a probe oligonucleotide hybridize to its target nucleic acid sequence in a diagnostic assay.
Goodchild, J., Abstract in Third International Symposium On Catalytic RNAs (Ribozymes) And Targeted Gene Therapy For The Treatment Of HIV Infection, San Diego Calif. (1992), describes the use of "facilitators" to improve the performance of hammerhead ribozymes. Facilitators are oligoribonucleotides that bind to a RNA substrate target sequence contiguously with a ribozyme binding site.
Tullis, U.S. Pat. No. 5,023,243, provides a general description of use of antisense oligonucleotides. Kaji, U.S. Pat. No. 4,689,320, provides data allegedly showing a decrease in mortality in mice infected with herpes simplex virus when treated with an antisense oligonucleotide having a nucleotide sequence targeted to herpes simplex virus. Goodchild et al., U.S. Pat. No. 4,806,463, provide data to demonstrate the ability of several different specified antisense oligonucleotides to inhibit HTLV-III (HIV) replication and gene expression in cultured cells infected with HIV. Cantin et al., U.S. Pat. No. 5,110,802, describe the use of a particular methylphosphonate-linked oligonucleotide to inhibit HIV replication (these U.S. patents are hereby incorporated by reference herein).
Matsukura et al., Proc. Natl. Acad. Sci. 86:4244, describe inhibition of HIV expression without killing the host cell using a phosphorothioate-linked oligonucleotide targeted to a rev sequence. Matsukura et al. also discuss using oligonucleotides joined by linkages other than phosphodiester or phosphorothioate groups. According to Matsukura et al., "certain chemically modified oligomers (e.g., methylphosphonates) have inherent limitations in that they are quite insoluble and require exceedingly high concentrations for biological effects."
Ribozymes, like antisense oligonucleotides, can be targeted to specific nucleic acid sequences based upon hybridization to a complementary nucleic acid sequence target site. The hybridized ribozyme can inhibit protein production from a target nucleic acid sequence by cleaving the nucleic acid sequence. Rossi et al., Aids Research And Human Retroviruses 8:183 (1992), in discussing using ribozymes as possible anti-HIV therapeutic agents, mention potential factors which may decrease accessibility of a target site. These factors may include HIV RNA having extensive secondary structure and cellular proteins associated with RNA.
Ribozymes and antisense oligonucleotides can be used to inhibit the functioning of nucleic acid sequences present in different organisms such as bacteria, plants, fungi, animals, or humans. For example, Fung et al., entitled "Gene therapy for cell proliferative diseases," PCT/US91/02478, discuss using ribozymes in humans to inhibit the functional expression of positive-acting growth regulatory elements to treat pathological cell proliferative diseases. Fung et al. also mention using antisense RNA as a means of suppressing gene expression.